CN101723890B - Aryl thiourea and preparation method and application thereof - Google Patents
Aryl thiourea and preparation method and application thereof Download PDFInfo
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- CN101723890B CN101723890B CN2009102061390A CN200910206139A CN101723890B CN 101723890 B CN101723890 B CN 101723890B CN 2009102061390 A CN2009102061390 A CN 2009102061390A CN 200910206139 A CN200910206139 A CN 200910206139A CN 101723890 B CN101723890 B CN 101723890B
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- 0 *c1cc(Oc(cc2)ccc2NC(Nc2ccncc2)=S)ccn1 Chemical compound *c1cc(Oc(cc2)ccc2NC(Nc2ccncc2)=S)ccn1 0.000 description 1
Abstract
The invention discloses aryl thiourea and a preparation method and applications thereof. The aryl thiourea is a compound shown in the general formula I. The new compound-aryl substituted thiourea shows very high activity of inhibiting the growth of tumor cells and especially has significant effect of inhibiting the growth of HUVEC cells with high expression of VEGFR, wherein IC 50 is more than 70% in 10mu g/ml. The invention also provides a preparation method of the compound.
Description
Technical field
The present invention relates to aryl thiourea new compound, synthetic and suppress the purposes of VEGFR-2, more particularly, is to contain the aryl thiourea compound as antitumour drug and synthetic and the purposes of target VEGFR efficiently, belongs to pharmaceutical field.
Background technology
Vascular endothelial growth factor receptor (Vascular Endothelial Growth FactorReceptor, VEGFR) Tyrosylprotein kinase is because of its keying action in mediation tumor-blood-vessel growth process, be antitumor drug design target [Sebolt-Leopold, J.S. at present of greatest concern; English, J.M., Mechanisms of drug inhibition of signalling molecules.Nature2006,441, (7092), 457-62].This growth factor receptors family has now found three regulation and control transmembrane glycopeptide polymeric immunoglobulin receptors [Neufeld, G.; Cohen, T.; Gengrinovitch, S.; Poltorak, Z., Vascular endothelial growth factor (VEGF) and its receptors.FASEB J1999,13, (1), 9-22.]: VEGFR-1 (Fms-like Tyrosine Kinase, Flt-1), VEGFR-2 (Kinase Insert Domain Containing Receptor, KDR/Fsk-1) and VEGFR-3.As other protein kinase, they shift the ATP terminal phosphate by catalysis, the cell growth signals are transferred on the tyrosine residues of target protein matrix, are regulating and control many vital processes thus, as cell proliferation, and growth, transfer and apoptosis etc.
VEGFR-2 is the single transmembrane protein, and is the same with all VEGFR, is divided into the outer ligand binding domain of film, strides the tyrosine kinase domain in film district and the film.The film outskirt structural flexibility of VEGFR-2 is big, and its conformational change of front and back is also big combining with VEGF, is not the ideal role target spot of micromolecular compound therefore.Tyrosine kinase domain is divided into several important function district [Crystal structure of the kinasedomain of human vascular endothelial growth factor receptor 2:a keyenzyme in angiogenesis.Structure 1999 in the VEGFR-2 film, 7, (3), 319-30.]: the A) hinge region of two albumen leaflets of connection; B) between two leaflets flat Triphosaden (AdenosineTri-Phosphate is ATP) in conjunction with the territory; C) be rich in the nucleosides coupling collar of glycine, the catalysis ring, the activation ring of highly flexible and kinases insert the territory.Micromolecular inhibitor combines with enzyme competitively with ATP, stops the activation of enzyme, thereby the signal transduction pathway of blocking VEGF reaches the purpose of the vasculogenesis that suppresses the VEGF mediation.Therefore, the ATP of the interior tyrosine kinase domain of VEGFR-2 film becomes the binding site of ideal micromolecular inhibitor naturally in conjunction with the territory.
So far, the inhibitor research of VEGFR-2 has obtained bigger progress.Xarelto (nexavar, sorafenib) be by the exploitation of U.S. Onyx drugmaker and German Bayer AG at VEGFR-2, VEGFR-3, many target spots antitumor drug of signal pathways such as RAF/MEK/ERK can suppress the propagation of tumour cell and the generation of blood vessel during it is tested effectively before clinical.In testing in the clinical III phase of metastatic renal cell cancer, Xarelto has improved patient's total lifetime of [Preclinicaloverview of sorafenib significantly, a multikinase inhibitor that targets both Rafand VEGF and PDGF receptor tyrosine kinase signaling.Mol Cancer Ther2008,7, (10), 3129-40.].Another TKI medicine that can act on VEGFR-2 is the Sutent (sutent of Pfizer, sunitinib), in treating in clinical III phase at random, Sutent has successfully suppressed imatinib is produced the gastrointestinal stromal tumor patient's of resistance tumor development [Efficacyand safety of sunitinib in patients with advanced gastrointestinalstromal tumour after failure of imatinib:a randomised controlledtrial.Lancet 2006,368, (9544), 1329-38.].
Therefore at present existing medicine still can not satisfy the demand of clinical application fully, and medicament research and development person is also seeking the new inhibiting compound of VEGFR-2 that has energetically.Anthranilamide pyridinureas compounds as the VEGFR kinase inhibitor is disclosed as CN101056871A, CN101052634A.CN1972925A discloses the two Arylurea derivatives that a class has the VEGFR kinase inhibitory activity, but the multiple group compound of substituting group definition is never implemented in the general formula, and with disclosed substituting group compound bigger nature difference is arranged, so we can't predict the thiourea that comprises in the general formula and whether have VEGFR kinase inhibiting activity and safety of medicine performance thereof.
Summary of the invention
Technical scheme of the present invention provides a class and has the Arylthioureas new compound that suppresses the conduction of vascular endothelial growth factor receptor signal.Technical scheme of the present invention provides the Preparation method and use of a class aryl thiourea compound.
A class aryl thiourea compound provided by the invention has following structure I
Wherein, R is C
6-10Aryl, C
4-C
8Heteroaryl, the C of replacement
6-10Aryl, the C of replacement
4-C
8Heteroaryl, described C
6-10Aryl or C
4-C
8Heteroaryl is replaced by one or more following groups :-CN ,-CF
3,-NO
2,-CO
2R
1,-C (O) NR
1R
1' ,-OR
1,-SR
1, halogen or perhalogeno at the most;
R
1And R
1' independently be selected from: H, C
1-10Alkyl, C
6-14Aryl, C
3-13Heteroaryl, the C of perhalogeno at the most
1-10Alkyl.
Compound of the present invention preferably from:
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
Compound 13
Compound 14
Compound 15
Compound 16
Shown in the synthetic following reaction formula of formula I compound of the present invention:
Specifically, with different sulphur nitrile acid esters (II) and [4-(4-amino-benzene methoxy base) (2-pyridyl)]-N-methylformamide (III) be a raw material, the direct condensation of mixing obtains the aryl thiourea derivative in solvent.Wherein, solvent is selected from: acetonitrile, ethyl acetate, methyl alcohol, ethanol, N, dinethylformamide (DMF) and dimethyl sulfoxide (DMSO) (DMSO).Temperature of reaction is 20-100 ℃, generally at room temperature or under the reflux temperature of solvent reacts.[4-(4-amino-benzene methoxy base) (2-pyridyl)]-N-methylformamide (III) with the material molar ratio of different sulphur nitrile acid esters (II) is: 1: 0.8-1.2.
Formula I compound of the present invention is as the purposes of the inhibitor of VEGFR, preferably its purposes in the preparation antitumor drug.Described tumour includes but not limited to: colorectal carcinoma, duodenal cancer, prostate cancer, mammary cancer, melanoma, duct carcinoma, liver cancer, carcinoma of the pancreas, kidney, carcinoma of endometrium, cancer of the stomach, nonsmall-cell lung cancer, neural malignant tumour and hematologic malignancies.
We have synthesized a large amount of novel series compounds by the design of computer molecular model.Experimental studies have found that by a large amount of in-vitro screenings these a series of aryl thiourea compounds provided by the invention have significant inhibition VEGFR-2 and antineoplastic activity, and its effect significantly is better than carbamide compounds sorafenib.In addition, by investigating influence to normal hepatocyte growth, we are surprised to find that also aryl thiourea compound provided by the invention will be significantly less than carbamide compounds sorafenib to the influence of normal hepatocyte growth, that is to say that aryl thiourea compound provided by the invention has more hypotoxicity to people's normal liver cell, show its better antitumor drug safety performance.
The present invention is based on the VEGFR protein kinase and small molecules suppresses interactional crystalline structure [Harris, P.A.; Cheung, M.; Hunter, R.N., 3rd; Brown, M.L.; Veal, J.M.; Nolte, R.T.; Wang, L.; Liu, W.; Crosby, R.M.; Johnson, J.H.; Epperly, A.H.; Kumar, R.; Luttrell, D.K.; Stafford, J.A., Discoveryand evaluation of 2-anilino-5-aryloxazoles as a novel class of VEGFR2kinase inhibitors.J Med Chem 2005,48, (5), 1610-9.], synthetic method by the focus type compound library, selected is core texture with the pharmacophore aryl thiourea, by on the thiocarbamide nitrogen-atoms, introducing different substituting groups, synthetic a series of different substituted aryl thiocarbamide new compounds, measured the inhibition activity of each compound at molecular level to the vascular endothelial growth factor receptor protein kinase, simultaneously measured the inhibition activity of each compound, and proved the restraining effect of The compounds of this invention tumour by experimentation on animals to the growth of tumour cell of vascular endothelial growth factor receptor overexpression at cell levels.
Embodiment
Below in conjunction with embodiment the present invention is further described, but is not construed as limiting the invention.
Synthesizing of embodiment 1 intermediate III
Step 1:
In the 250mL there-necked flask, logical nitrogen replacement air, and in N
2Protection adds 36mLSOCl down
2, carefully splash into 1.2mL exsiccant DMF, 42 ℃ of temperature controls, after stirring 10min, slowly add 12g 2-pyridine carboxylic acid (adding in the 30min), be warming up to 72 ℃ then, stirring reaction 16h is cooled to room temperature, adds 100mL toluene, be evaporated to 40mL, be concentrated into 40mL after adding 60mL toluene again, repeat said process once, have a small amount of red solid to occur, suction filtration, with 50mL toluene wash solid, filtrate places the 250mL flask, and ice-water bath slowly adds 40mL methyl alcohol down, stir 10min, remove ice bath, stir 45min under the room temperature, place ice-water bath 5min again, add the 40mL ether, the adularescent precipitation is separated out, and filters, with 30mL ether washing gained solid, get pale yellow powder (2) 14.9g, productive rate 73.5% after the drying.
Step 2:
In the 250mL there-necked flask, compound (2) 14.9g is dissolved in the mixed solvent of 10mL methyl alcohol and 50mL THF, N
2Protect following 0 ℃ to stir 10min, slowly splash into the 70mL methylamine alcohol solution, keep temperature of reaction to be lower than 5 ℃, drip and finish; behind the reaction 5h, the evaporated under reduced pressure solvent adds 150mL ethyl acetate debris under 0 ℃; filter, with 50mL ethyl acetate washing gained filter residue, gained filtrate anhydrous Na
2SO
4After the drying, revolve steam yellow oily liquid (3) 12.4g.
Step 3:
Add the 8.8g p-aminophenol in the 250mL flask, 130mL dry DMF and 8.5g potassium tert.-butoxide behind the stirring 2h, add the 5.4g anhydrous K under the room temperature
2CO
3And 12.4g compound (3), N
2Be warming up to 80 ℃ of reaction 6h under the protection, add ethyl acetate and saturated Na in the reaction solution
2CO
3The solution separatory, organic layer is washed with saturated common salt, anhydrous Na
2SO
4After the drying, silica gel column chromatography separates (PE: EA=2: 1 (v/v)) get the pale brown look solid of 6.0g (III).
1HNMR(600MHz，CDCl3)：δ3.00(d，3H)，3.69(br?s，2H)，6.71(d，2H)，6.87(d，2H)，6.90-6.91(m，1H)，7.66(d，1H)，7.99(br?s，1H)，8.32(d，1H).
Synthesizing of the different sulphur nitrile of embodiment 2 intermediate II acid esters
Method 1:
Add the 1.85g P-nethoxyaniline in the 100mL single port bottle, 6.3mL triethylamine and 20mL toluene under the stirring at room, drip 2.7mL CS in 20min
2, add the back and continue room temperature reaction until separating out a large amount of solids.Ice-water bath is the above-mentioned reaction solution of cooling down, slowly drip the 15mL toluene solution that is dissolved with 1.48g three surpalites (BTC), add the back and continue the about 4h of room temperature reaction, filter, filter residue is washed 2 times with toluene, filtrate decompression distill crude product, silica gel column chromatography separate (pure PE) colourless liquid II-1 1.86g, productive rate 75.1%.
1HNMR(600MHz，CDCl3)：δ3.79(s，3H)，6.83-6.84(d，2H)，7.13-7.15(d，2H).
Method 2:
Add 0.89g in the 100mL single port bottle to trifluoro-methoxyaniline, 3.30g triethylene diamine six water and 10mL toluene under the stirring at room, drip 0.9mL CS in 20min
2(being dissolved in 15mL toluene) adds the back and continues room temperature reaction until separating out a large amount of solids.Suction filtration, and with small amount of toluene washing gained solid, dry yellow solid.Above yellow solid is suspended in the 15mL methylene dichloride, ice-water bath is cooled to 5-10 ℃, slowly drip the 15mL dichloromethane solution that is dissolved with 0.50g three surpalites (BTC), add back room temperature reaction 1h, temperature rising reflux reacts completely then, is cooled to room temperature, remove by filter insolubles, filtrate decompression distill crude product, silica gel column chromatography separate (pure PE or PE: EA=5: 1 (v/v)) colourless liquid II-20.56g, productive rate 50.7%.
1HNMR(600MHz，CDCl3)：δ7.18-7.20(d，2H)，7.23-7.24(d，2H).
The preparation of embodiment 3 compounds 1
In the 25mL single port bottle, the 73mg intermediate III is dissolved among the 5mL DMF, drip under the ice-water bath cooling and stirring be dissolved among the 5mL DMF to the different sulphur nitrile of trifluoromethoxy benzaldehyde acid esters 71mg, drip complete room temperature reaction, after TLC plate detection reaction is finished, use ethyl acetate extraction, the saturated common salt washing, anhydrous Na
2SO
4After the drying, silica gel column chromatography separates (PE: EA=2: 1 (v/v)) get the light grey powder of 76mg, productive rate 52.8%.
1H?NMR(600MHz，DMSO-d6)：δ2.96-2.97(d，3H)，7.01-7.02(m，1H)，7.05-7.06(d，2H)，7.18-7.19(d，2H)，7.42-7.45(t，4H)，7.62(d，1H)，8.11-8.12(br?d，1H)，8.39-8.40(d，1H)，8.64(s，1H)，8.70(s，1H).
MS(ESI)：461.06(M-H)
The preparation of embodiment 4 compounds 2
Working method is with embodiment 3, and used intermediate II is that the molar ratio of intermediate III and intermediate II is 1: 0.8 to the different sulphur nitrile of fluorobenzene acid esters.Solvent for use is DMF, 50 ℃ of temperature, and productive rate is 86%.
1H?NMR(600MHz，DMSO-d6)：δ2.91(d，3H)，6.90(d，2H)，6.98(d，2H)，7.02(m，1H)，7.07(m，1H)，7.31(d，2H)，7.55(d，2H)，8.05(br，3H)，8.51(m，1H).
MS(ESI)：395.02(M-H)
The preparation of embodiment 5 compounds 3
Working method is with embodiment 3, and used intermediate II is to the different sulphur nitrile of bromobenzene acid esters, and the molar ratio of intermediate III and intermediate II is that 1: 1.2 solvent for use is DMF, 20 ℃ of temperature, and productive rate is 67%.
1H?NMR(600MHz，DMSO-d6)：δ2.99-3.00(d，3H)，7.02-7.03(m，1H)，7.10-7.12(t，4H)，7.36-7.38(m，2H)，7.44-7.45(d，2H)，7.67(d，1H)，7.85(s，1H)，7.87(s，1H)，8.01(br?d，1H)，8.41-8.42(d，1H).
MS(ESI)：456.34(M-H)
The preparation of embodiment 6 compounds 4
Working method is with embodiment 3, used intermediate II be between the different sulphur nitrile of ethyl benzoate acid esters, the molar ratio of intermediate III and intermediate II is 1: 1.0, solvent for use is DMF, 20 ℃ of temperature, productive rate is 75%.
1HNMR(600MHz，DMSO-d6)：δ1.36(t，3H)，2.98(s，3H)，4.36(q，2H)，6.90(dd，2H)，7.02(m，1H)，7.01(d，1H)，7.31(dd，2H)，7.45(m，1H)，7.74(m，1H)，7.95(d，1H)，8.16(d，1H)，8.39(br，3H)，8.51(d，1H).
MS(ESI)：450.51(M)
+
The preparation of embodiment 7 compounds 5
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 1-naphthalene acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.2, and solvent for use is DMSO, 100 ℃ of temperature, and productive rate is 86%.
1HNMR(600MHz，DMSO-d6)：δ2.75-2.76(d，3H)，7.10-7.12(m，1H)，7.14-7.16(d，2H)，7.38(d，1H)，7.50-7.55(m，4H)，7.59-7.60(d，2H)，7.83-7.85(t，1H)，7.93-7.97(m，2H)，8.48(d，1H)，8.71(br，1H)，9.78(s，1H)，9.89(s，1H).
MS(ESI)：429.22(M+H)
+
The preparation of embodiment 8 compounds 6
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 6-indoles acid esters, and the molar ratio of intermediate III and intermediate II is 1: 0.8, and solvent for use is an ethyl acetate, 70 ℃ of temperature, and productive rate is 54%.
1HNMR(600MHz，DMSO-d6)：δ2.76-2.77(d，3H)，6.45(s，1H)，7.04-7.07(t，1H)，7.11-7.12(m，1H)，7.13-7.14(d，2H)，7.22-7.25(t，2H)，7.32-7.33(t，1H)，7.39(d，1H)，7.60-7.62(d，2H)，8.49-8.50(d，1H)，8.72(br?d，1H)，9.61(s，1H)，9.84(s，1H)，11.16(br?s，1H).
MS(ESI)：416.20(M-H)
+
The preparation of embodiment 9 compounds 7
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 1-pyrroles acid esters, and the molar ratio of intermediate III and intermediate II is 1: 0.8, and solvent for use is an ethanol, 20 ℃ of temperature, and productive rate is 48%.
1HNMR(600MHz，DMSO-d6)：δ2.98(s，3H)，5.97(d，1H)，6.23(m，1H)，6.90(d，2H)，6.96(d，1H)，7.02(m，1H)，7.07(d，1H)，7.35(dd，2H)，8.51(d，1H)，9.52(br，4H).
MS(ESI)：367.41(M)
+
The preparation of embodiment 10 compounds 8
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 3-benzoyl methylamine acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.0, and solvent for use is DMF, 20 ℃ of temperature, and productive rate is 84%.
1H?NMR(600MHz，DMSO-d6)：δ2.82(s，3H)，2.98(s，3H)，6.90(dd，2H)，7.02(m，1H)，7.07(d，1H)，7.31(dd，2H)，7.68(m，1H)，7.76(m，1H)，7.82(m，1H)，8.01(br，4H)，8.13(d，1H)，8.51(d，1H).
MS(ESI)：436.49(M+H)
+
The preparation of embodiment 11 compounds 9
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 4-benzanilide acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.2, and solvent for use is DMF, 100 ℃ of temperature, and productive rate is 78%.
1HNMR(600MHz，DMSO-d6)：δ2.91(s，1H)，6.90(dd，2H)，7.02(m，1H)，7.07(d，1H)，7.14(m，1H)，7.31(dd，2H)，7.39(m，2H)，7.86(m，2H)，7.89(dd，2H)，8.16(dd，2H)，8.38(br，4H)，8.51(d，1H).
MS(ESI)：498.56(M+H)
+
The preparation of embodiment 12 compounds 10
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 4-thioanisole acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.2, and solvent for use is DMF, 50 ℃ of temperature, and productive rate is 79%.
1HNMR(600MHz，DMSO-d6)：δ2.60(s，3H)，2.91(s，3H)，6.90(dd，2H)，7.02(m，1H)，7.07(d，1H)，7.30(dd，2H)，7.31(dd，2H)，7.51(dd，2H)，8.05(br，3H)，8.51(d，1H).
MS(ESI)：424.53(M)
+
The preparation of embodiment 13 compounds 11
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 4-pyridine acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.0, and solvent for use is DMF, 20 ℃ of temperature, and productive rate is 62%.
1HNMR(600MHz，DMSO-d6)：δ2.91(s，3H)，6.90(dd，2H)，7.02(m，1H)，7.07(d，1H)，7.31(dd，2H)，7.80(dd，2H)，8.05(br，3H)，8.45(dd，2H)，8.51(d，1H).
MS(ESI)：397.75(M)
+
The preparation of embodiment 14 compounds 12
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 3-oil of mirbane acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.1, and solvent for use is an acetonitrile, 50 ℃ of temperature, and productive rate is 87%.
1HNMR(600MHz，DMSO-d6)：δ2.91(s，3H)，6.90(dd，2H)，7.02(m，1H)，7.07(d，1H)，7.31(dd，2H)，7.67(m，1H)，7.86(m，1H)，8.05(br，3H)，8.20(m，1H)，8.51(d，1H)，8.87(d，1H).
MS(ESI)：422.44(M-H)
+
The preparation of embodiment 15 compounds 13
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 2-itrile group benzene acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.2, and solvent for use is DMF, 20 ℃ of temperature, and productive rate is 79%.
1HNMR(600MHz，DMSO-d6)：δ2.91(s，3H)，6.90(dd，2H)，7.02(m，1H)，7.07(d，1H)，7.22(m，1H)，7.31(dd，2H)，7.54(m，1H)，7.66(m，1H)，7.75(m，1H)，8.07(br，3H)，8.51(d，1H).
MS(ESI)：402.45(M-H)
+
The preparation of embodiment 16 compounds 14
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 4-chloro-3-trifluoromethylbenzene acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.0, and solvent for use is an acetonitrile, 30 ℃ of temperature, and productive rate is 85%.
1H?NMR(600MHz，DMSO-d6)：δ2.97-2.98(d，3H)，7.05-7.06(m，1H)，7.07-7.08(d，2H)，7.41-7.44(t，3H)，7.60(d，1H)，7.66-7.68(dd，1H)，7.71(d，1H)，8.14-8.15(br?d，1H)，8.41-8.42(d，1H)，8.61(s，1H)，8.81(s，1H).
MS(ESI)：479.04(M-H)
+，480.10(M
+)
The preparation of embodiment 17 compounds 15
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a 5-chloronaphthalene acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.0, and solvent for use is DMF, 20 ℃ of temperature, and productive rate is 65%.
1HNMR(600MHz，DMSO-d6)：δ2.91(s，3H)，6.90(dd，2H)，7.02(m，1H)，7.07(d，1H)，7.31(dd，2H)，7.39(m，1H)，7.44(m，1H)，7.71(m，1H)，7.78(m，1H)，8.05(m，1H)，8.05(br，3H)，8.09(m，1H)，8.51(d，1H).
MS(ESI)：462.95(M)
+
The preparation of embodiment 18 compounds 16
Working method is with embodiment 3, and used intermediate II is the different sulphur nitrile of a benzene acid esters, and the molar ratio of intermediate III and intermediate II is 1: 1.0, and solvent for use is DMF, 20 ℃ of temperature, and productive rate is 91%.
1HNMR(600MHz，DMSO-d6)：δ2.91(s，3H)，6.91(dd，2H)，7.02(m，1H)，7.07(d，1H)，7.06(m，1H)，7.31(dd，2H)，7.34(m，2H)，7.58(dd，2H)，8.05(br，3H)，8.51(d，1).
MS(ESI)：378.45(M)
+
Below prove beneficial effect of the present invention by the concrete test of pesticide effectiveness.
Test example 1 The compounds of this invention is tested vascular endothelial growth factor receptor [VEGFR] protein kinase inhibiting activity:
Cytoplasmic region sequence Val 805-Val 1356 great expression in the Sf9 cell with the VEGFR-2 protein kinase, be inoculated in 6 orifice plates (5 ' 105/ hole), after the serum-free medium synchronization 24 hours, under 4 ℃ with cell pyrolysis liquid effect 20 minutes, scrape cell, the ultrasonication cell, supernatant is collected in centrifugal back, sends out by continuous chromatography and separates the kinase protein that obtains purity＞90%.
In 96 orifice plates, use poly (Glu4-Tyr) proportioning in Tris buffered soln (25mM Tris, pH7.2,150mM NaCl) to become the suspension of 0.5mg/mL, 1 as a child used twice of TBS solution washing.The VEGFR-2 kinase dilution adds with the compound solution of DMSO preparation and hatched 10 minutes in kinase reaction buffered soln.In each hole, add ATP/MgCl
2Provocative reaction, last strength of solution are formed: the ATP of 1M, the MgCl of 10mM
2, the MnCl of 1mM
2, the HEPES of 4mM, pH are 7.4, the Na of 20mM
3VO
4, the BSA of 20mg/mL and 2% DMSO.After following 40 minutes of the room temperature, remove clear liquid, and wash 3 times with TBST solution (TBS with 0.05% Tween 20).The europium conjugated anti phosphotyrosine antibody 75ml that adds the about 0.1mg/mL of concentration was hatched 1 hour, used the TBST solution washing then 3 times, added 100ml enhancing liquid and hatched in camera bellows 15 minutes.Then 96 orifice plates are placed many labels of dimension gram multivoltage sales counter to use the europium detection protocol of acquiescence to carry out reading.Compare the inhibition percentage that calculates compound with blank sample data.
Table 1 compound 1-16 is to the inhibition effect of VEGFR protein kinase
The enzymic activity test of molecular level shows that aryl thiourea compound provided by the invention has very strong inhibition effect to the vascular endothelial growth factor receptor protein kinase, and its inhibiting rate is significantly higher than sorafenib, and particularly compound 2,4,5,6,9,10,14 and 16.
Test example 2 The compounds of this invention suppress active testing to the growth of tumour cell of vascular endothelial growth factor receptor [VEGFR] protein kinase high expression level:
Human umbilical vein endothelial cells [HUVEC] high expression level vascular endothelial growth factor receptor is used for the inhibition effect of test compounds to growth of tumour cell.
Observe the proliferation function of compound with tetrazolium (MTT) method: with the HUVEC of logarithmic phase for cell, use 0.25% tryptic digestion, be made into the individual cells suspension with the 1640 culture medium culturing liquid that contain 10% tire calf serum, with 104 cell inoculations in every hole in 96 orifice plates, every hole 100mL is hatched 24h in incubator.Under aseptic condition, handled 96 well culture plates (every kind compound establish 3 parallel) 24-72 hour with the pastille nutrient solution of no medicine nutrient solution and various concentration.Cultivate and finish preceding 4 hours adding MTT colour developing, can form coloured crystallization in viable cell, the centrifugal back of suspension cell is inhaled and is abandoned supernatant liquor.With DMSO dissolving crystallized after, measure the absorbancy at 570nm place.Try to achieve inhibiting rate (%) by the following method.
Proliferation inhibition rate=1-(experimental port A value-blank well A value)/(control wells A value-blank well A value)
Table 2 compound 1-16 is to the inhibition effect of Human umbilical vein endothelial cells [HUVEC] growth
The pharmacology result of cell levels shows, aryl thiourea compound provided by the invention is the highly active tumor growth inhibitor of a class, tumour cell to the vascular endothelial growth factor receptor high expression level shows very high inhibition activity, significantly be better than sorafenib, especially compound 6,9,11,12 and 14.
Test of the effect of example 3 The compounds of this invention to liver cancer:
It is 5 * 10 that the liver cancer SMMC-7721 cell of logarithmic phase growth is made density with phosphoric acid buffer (PBS)
7The cell suspension of individual/ml, getting 0.2ml, to be inoculated in the right armpits of 70 BALB/C nude mices subcutaneous.Behind 8d, be divided into 2,6,9,11,14 groups of model group, control group and compounds at random.Oral administration, experimental group give the compound 2,6,9,11,14 of 0.1g/kg body weight respectively, every day 1 time, give the physiological saline of model group equivalent, the sorafenib of control group 0.1g/kg body weight equally.4 weeks back execution animal is taken out the knurl piece under the sterile state, measure major diameter, the minor axis of transplanted tumor, and claim knurl heavy.Gross tumor volume (V)=(major diameter * minor axis)
2/ 2, volume tumour inhibiting rate=(control group volume-experimental group volume)/control group volume * 100%; Weight tumour inhibiting rate=(control group knurl weight-experimental group knurl is heavy)/control group knurl heavy * 100%.Tumor microvessel density (MVD) press the Wendner method (Zhang Bixiang etc., COX-2 and vascular endothelial growth factor coexpression and the angiopoietic relation of hepatocellular carcinoma, Chinese experimental surgery impurity, 2004,21:671-675) detect.
Table 3 compound is to the experimental result of liver cancer effect
| Group | Volume tumour inhibiting rate (%) | Weight tumour inhibiting rate (%) | MVD |
| Model group | 0 | 0 | 20.94±5.63 |
| Control group | 61.32 | 59.28 | 9.64±1.91* |
| Compound 2 | 74.39 | 72.94 | 7.88±1.79*△ |
| Compound 6 | 79.54 | 76.52 | 7.54±1.97*△ |
| Compound 9 | 78.63 | 76.98 | 7.42±2.10*△ |
| Compound 11 | 76.58 | 73.43 | 7.73±1.86*△ |
| Compound 14 | 83.56 | 81.64 | 6.81±2.10*△ |
Annotate *: compare P＜0.01 with model group
△: compare P＜0.05 with control group
Zooperal result shows that aryl thiourea compound provided by the invention and sorafenib all can significantly suppress growth of tumor, significantly reduce MVD, has confirmed that the two all has the effect that tumor vessel forms that suppresses.Compare with sorafenib, the tumour inhibiting rate of compound 2,6,9,11,14 significantly improves, and the effect that reduces animal pattern MVD also significantly strengthens (P＜0.05).
Embodiment 4: the influence that medicine is bred human body normal liver cell LO-2:
People's normal liver cell LO-2 that will be in logarithmic phase is with behind the tryptic digestion of 1mI 0.5%, and the wave and culture bottle makes Digestive system flow through all cell surfaces gently.At room temperature hatch about 8min, culturing bottle is placed under the inverted microscope observes, after discovery kytoplasm retraction, intercellular substance increase, stop digestion immediately.Outwell Digestive system in the culturing bottle, add the nutrient solution that contains bovine serum, blow and beat a bottle tapetum cell repeatedly, make cell suspension with the elbow suction pipe.Adjusting concentration of cell suspension with the RPMI RPMI-1640 that contains 10% foetal calf serum is 1.0 * 10
5Individual/L.Get the 190uL cell suspension inoculation in 96 well culture plates, cultivate 12h in 37 ℃, 5%CO2 incubator.After treating cell attachment, in sample well, add 10ul Xarelto, compound 2, compound 6, compound 9, compound 11 and compound 14 respectively, all establish 6 parallel holes for every group.The blank group adds the equal-volume nutrient solution.Behind drug effect 24,48,72h, it is the four tetrazolium bromide solution of 5mg/mL that every hole adds 10ul concentration, 37 ℃, 5%CO
2After continuing in the incubator to hatch 4h, every hole adds DMSO150ul, and middling speed vibration 15s with the zeroing of blank hole, detects each sample well absorbancy (D), twice of every hole replication with microplate reader at the 570nm place.Proliferation inhibition rate (%)=(D contrast-D mensuration)/D contrast * 100%.
Table 4 compound is to the influence of people's normal liver cell LO-2 proliferation inhibition rate
Annotate *: compare P＜0.05 with the sorafenib group;
*: compare P＜0.01 with the sorafenib group
The result shows: each group all shows in various degree cell growth inhibiting effect to people's normal liver cell LO-2, and along with the prolongation of action time, restraining effect presents enhanced trend.After 24 hours, the proliferation inhibition rate of compound 6,9,11 and compound 14 is starkly lower than sorafenib control drug (P＜0.05 or P＜0.01) at drug effect.Act on after 48,72 hours, the cyto-inhibition of all compounds all significantly is lower than sorafenib (P＜0.05 or P＜0.01).
Therefore, prove that by experiment in vivo and vitro aryl thiourea compound provided by the invention is highly active new vascular endothelial growth factor receptor protein kinase growth inhibitor of a class and tumor growth inhibitor, and (people's normal liver cell LO-2) has significant low cytotoxicity to the human body normal liver cell, shows its better antitumor drug safety performance.
Claims (8)
1. a class Arylthioureas compounds, general structure is:
Wherein R is C
6-10Aryl, C
4-C
8Heteroaryl, the C of replacement
6-10The C of aryl or replacement
4-C
8Heteroaryl; Described C
6-10Aryl or C
4-C
8Heteroaryl is replaced by one or more following groups :-CN ,-CF
3,-NO
2,-CO
2R
1,-C (O) NR
1R
1' ,-OR
1,-SR
1Or halogen;
Wherein, R
1And R
1' be: H, C
6-14The aryl or the C of perhalogeno at the most
1-10Alkyl.
2. the described Arylthioureas compounds of claim 1, it is characterized in that: described compound is:
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
Compound 13
Compound 14
Compound 15
Compound 16.
3. the preparation method of claim 1 or 2 described Arylthioureas compounds is characterized in that: formula II compound and formula III compound are mixed direct condensation obtain formula I in solvent:
4. the preparation method of Arylthioureas compounds according to claim 3, it is characterized in that: employed solvent is: acetonitrile, ethyl acetate, methyl alcohol, ethanol, N, dinethylformamide or dimethyl sulfoxide (DMSO).
5. the preparation method of Arylthioureas compounds according to claim 3, it is characterized in that: temperature of reaction is 20-100 ℃.
6. the preparation method of Arylthioureas compounds according to claim 3, it is characterized in that: the mol ratio of formula III compound and formula II compound is 1: 0.8-1.2.
7. claim 1 or the 2 described compounds application in the medicine of the disease that the preparation treatment is caused, mediates and/or propagated by the vascular endothelial growth factor receptor kinases.
8. application according to claim 7 is characterized in that: described disease is a tumour.
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| CN103319402B (en) * | 2012-03-22 | 2015-01-14 | 齐鲁制药有限公司 | Diphenyl thiourea derivatives, preparation method and applications thereof |
| CN102653522B (en) * | 2012-04-13 | 2015-12-02 | 烟台大学 | Diphenyl thiourea compounds of ω-carboxyl substituted and its production and use |
| CN102993093A (en) * | 2012-09-27 | 2013-03-27 | 烟台大学 | N,N'-2-substitubted-diphenylthiourea compound, as well as preparation method and applications thereof |
| CN104710355B (en) * | 2015-02-15 | 2017-06-16 | 烟台大学 | The preparation method and purposes of diphenyl thiourea compounds and its salt of one class containing niacinamide building block |
| CN105753841B (en) * | 2016-01-18 | 2018-01-05 | 西安交通大学 | A kind of N indazoles substituting thioureido analog derivative and its preparation method and application |
| US10519113B2 (en) * | 2016-08-17 | 2019-12-31 | Icahn School Of Medicine At Mount Sinai | Kinase inhibitor compounds, compositions, and methods of treating cancer |
| CN106543077B (en) * | 2016-10-27 | 2019-08-23 | 西安交通大学 | N, N '-disubstituted arylthiourea derivative and its synthetic method and application |
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